JPH10136988A - Recombinant pseudomonas exotoxin: structure of active immunotoxin having less side effect - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an expression plasmid containing a DNA encoding a complex compound containing a modified Pseudomonas exotoxin having an activity of an ADP ribosylation and a capability of a translocation mediated by a cell membrane. SOLUTION: This recombinant Pseudomonas exotoxin is obtained by using an expression plasmid containing a DNA encoding a complex compound containing a modified Pseudomonas exotoxin having an activity of an ADP ribosylation and a capability of a translocation mediated by a cell membrane. The modified exotoxin has a low toxicity to human or an animal in vitro and also has a modification at a receptor binding domain Ia of a natural toxin sufficient to become a modified toxin having a low toxicity to liver on administration in vivo, compared with those of an unmodified Pseudomonas exotoxin.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION Toxins cause extremely powerful cell destruction, which causes many human diseases. Due to its strong activity, toxins are conjugated to monoclonal antibodies to make cytotoxic agents (immunotoxins) that specifically bind to target cells. Therefore, these immunotoxins are most beneficial in cancer therapy.

[0002] Pseudomonas exotoxin A (PE)
Is an extremely active protein monomer (molecular weight 66K
d) and Pseudomonas aer
uginosa). This protein monomer catalyzes ADP-ribosylation of peptide chain elongation factor 2 (EF-2) (catalyzes the transfer of the ADP-ribose moiety of oxidized NAD ⁺ to EF-2), thereby realizing EF2 inactivation. It inhibits protein synthesis in nuclear cells.

The process of poisoning is thought to be as follows. : First, PE binds with a specific receptor on the cell surface. Next, the PE-receptor complex is absorbed into the cell.
Eventually, PE enters the cytosol where it enzymatically inhibits protein synthesis. NH that raises the pH of acidic vacuoles
The 4 ⁺ such weak base, poisoning the cells are prevented, the transition process is believed to occur from an acidic zone. The hydrophobic domain of PE enters the cell membrane upon exposure to the acidic bear and forms a pathway through the cytosol with the enzyme domain extended.

[0004] US Patent No. 4,545,985 teaches that pseudomonastoxin can be chemically bound to antibodies or growth factors. However, these chemically conjugated toxins exhibit only undesirable levels of toxicity. Therefore, it would be beneficial to have toxins that act highly specifically to target certain cell types without the destruction of cells generally associated with these toxins.

[0005] Immunotoxins containing PE are derived from natural PE.
And iminothiolane. This reaction results in two new sulfhydryl groups that help bind the antibody to the toxin, and thus inactivates the binding of PE to its native receptor. This concept is based on chemically inactivating the PE binding site to minimize the undesirable side effects of PE binding to cells with PE receptors. This idea is quite good for destruction of specific cells in tissue cell culture medium and tumor-inoculated mice, but due to the toxic side effects of immunotokinin,
For a 20-gram mouse, 2 μg cannot be administered to a 3 Kg monkey, and 1 mg cannot be administered to an adult, and an adult cannot be administered more than 4 mg. It is therefore desirable to be able to administer larger amounts of immunotoxins in order to achieve greater destruction of tumor cells. The present invention achieves this goal by providing an immunotoxin with high efficacy and low toxicity. Furthermore, in order to overcome the above-mentioned idea of chemical inactivation of the PE binding site, a DNA recombination technique was used in the present invention. That is, DNA recombination techniques can be used to obtain a complete toxin gene (or a full-length toxin molecule (or a portion thereof) that contains domains of different function or that does not have a cell-binding domain). Or a fragment thereof) was cloned.
A comparative experiment of these clones is shown in Example 1.

[0006] The tertiary structure of PE has been determined by X-ray crystallography. As shown in FIG. 2, the PE molecule has three structurally different domains. Domain I consists of amino acid residues 1 to 252 (domain Ia), 365 to 404
(Domain Ib) contains; Domain II contains 253-364 amino acid residues; Domain III has 405-613 amino acid residues.

[0007] Plasmids have been produced which express various parts of the PE molecule. This plasmid allows the different structural domains of the PE molecule to be implicated in various functional activities, and (1) which parts of the molecule contribute to cell recognition (binding), (structural domain I, amino acids 1-252); (2) Which part is required for enzyme activity (ADP ribosylation activity, domain III plus domain Ib) (amino acids 385-61)
3); (3) allows to determine which parts contribute to translocation through the cell membrane (domain II).

The fact that the structural domain Ia is involved in cell recognition is clear from the following facts. That is, the protein produced by the plasmid containing domain II, domain Ib and domain III without domain Ia has no cytotoxicity of its own and does not show antagonistic inhibition of the cytotoxicity of the intact toxin. For domain I
Plasmids encoding a and not other domains blocked PE cytotoxicity of sensitive cells. PE obtained from a plasmid introduced by deleting the first half of structural domain II exhibits both blocking and ADP ribosylation activities. However, these molecules had lost all their cytotoxic activity. Plasmids encoding only horizontal domain III produce large amounts of protein, which has lost protective enzymatic activity (ADP-ribosylation).
However, plasmids encoding all of structural domain III and amino acids close to structural domain Ib express large amounts of proteins and have high ADP ribosylation activity.

[0009] Based on the tertiary structure of the PE, a plasmid is created such that different parts of the PE are expressed. The protein pattern of cells expressing different structures was analyzed by SDS gel electrophoresis, and the ADP ribosylation activity of the recombinant toxin was measured. Of these plasmids (described in Example 1), amino acids 253-613 (structural domain I
b, II, and III) and plasmid pJH17 containing the amino acids from domain Ib,
Large amounts of modified PE can be encoded that retain high enzymatic activity despite having low toxicity to human cells.

When the above plasmid patterns are combined,
Structural domain Ia is the receptor binding domain, the first half of structural domain II is the part required for translocation of toxin from the vacuole of host cell inclusions to the cytosol, and structural domain III is Alone does not show sufficient ADP-ribosylation activity.

When PE is administered to an animal, death occurs due to liver damage peculiar to PE. Similarly, since immunotoxins made from PE damage the liver, administration of large amounts of immunotoxins made from PE leads to death due to liver toxicity poisoning. The experiments shown in Table 3 show that structural domain Ia contributes to cell binding, as well as structural domain Ia.
PE molecules lacking a have been shown to be less toxic to mice than native PE. This conclusion is also supported by the data of Example 4, which shows that the toxicity of modified PE to mice is less than 200 times less than that of native PE. Example 5 shows that when a protein produced from pJH8 is purified and bound to an antibody against human transferrin receptor, it exhibits almost the same activity as an antitoxin containing natural PE, and cells other than target cells (mouse)
Toxicity indicates that immunotoxins as low as one hundredth are obtained.

Therefore, in other words, in other words, the PE molecule lacking the structural domain Ia has low liver toxicity.
It is an effective immunotoxin with few side effects.

[0013]

DETAILED DESCRIPTION OF THE INVENTION In a preferred embodiment; the invention is to produce a modified Pseudomonas exotoxin (PE) which upon modification becomes an active immunotoxin. The modified toxins and their immunotoxins produced therefrom have significantly reduced non-specific toxicity to human and mouse cells in tissue cell cultures, and significantly reduced toxicity in mouse in vivo cells.

[0014] Genomic DNA is obtained from EcoRI strain
And PstI for complete digestion. Although any strain of Pseudomonas aeruginosa is suitable for use in the present invention, PA103, which produces large amounts of active toxin, is the strain of choice. 2.6 ~
A 2.9 kb DNA fragment is isolated from the genomic DNA library. This fragment was digested with EcoRI and PstI.
It is ligated to a 2.7 kb long DNA fragment obtained by cutting the UC13 plasmid. The pUC13 plasmid is Bo
Available from ehringerMannheim. Suitable hosts are preferably E. coli.
(A preferred E. coli strain is HB101, which is Bethesd.
aResearchLaboratories).

Plasmid DNA is prepared from some positive colonies using ptoxETA, a 2.7 kb DNA fragment obtained from a plasmid containing the PE structural gene, as a probe, and identified by Southern blotting. The different sized modified PEs are then expressed in a suitable host. Sonozoic and [BL21 (D
E ₃ )] can be derived from bacteriophage T7RN
A host having the A polymerase gene is preferred, and this host is a Brookhaven National Labo.
ratories F. W. Available from Dr. Studier. Similarly, F. W. Bacteriophage late T7 promoter, AMP ^r and S, available from Dr. Studier
hine-Dalgarno box-pAR215
A plasmid with 6 is preferred. As shown in the examples, preferred plasmids are pJH8 and pJH17. p
JH8 is a 5.1 kb DNA fragment which contains the domain I of pE.
I, Ib, and III can be expressed. This plasmid is
It is produced by partially cutting plasmid pJH4 with AvaI. The linear DNA fragment is composed of AvaI and Hin.
It is cut completely with HindIII to obtain a 5.1 kb fragment with a recognition site for dIII.

This fragment is used to remove its sticky end,
S1 nuclease was added and incubated, and then ligated with T4 ligase to make plasmid pJH8.

The plasmid pJH17 has a 4.8 kb DN.
It contains the A fragment and is capable of expressing domain III with the 20 adjacent amino acids of PE. This plasmid is made by completely cutting plasmid pJH4 with ApaI and HindIII. The 4.8 kb DNA fragment is then separated, incubated with Klenow DNA polymerase I and dNTP to fill the sticky ends, and subsequently ligated with T4 ligase.

Expression of recombinant toxin in BL21 (DE ₃ ) BL containing plasmids expressing PEs of different sizes
21 (DE ₃ ) is 50 μg Ampicilli at 37 ° C.
Cultured in LB medium containing n / ml. When the absorbance reaches 0.3 at a wavelength of 650 nm, IPTG (isopro
pyl beta-D-thio-galatopyr
anoside) to the culture to a concentration of 1 mM. After 90 minutes the cells were harvested and the amount of recombinant toxin was determined by SD
Analyzed by S-PACE, immunoblot, ADP ribosylation, cytotoxicity experiments.

SDS-PAGE, immunoblotting Cell pellets are lysed in Laemmli buffer. The samples were boiled for 5 minutes before running on a 0.1% SDS, 10% acrylamide slab gel. In order to perform immunoblotting, the sample after electrophoresis is transferred to a nitrocellulose filter, and then reacted with an antibody against PE.
(Anti-rabbit-goat) and stain. PE
Are obtained from rabbits highly immunized with glutaraldehyde (0.2%) reacted with PE (inoculation of 250 μg of PE). For immunoblotting,
An IgG fraction is prepared.

ADP-ribosylation activity assay ADP-ribosylation activity assay was performed by a conventional method. Briefly, rabbit reticulocyte preparation or wheat germ extract enriched for peptide chain elongation factor 2 (EF-2) was used as EF-2 source. The assay solution (500 μl total) contains about 10 pmole of EF-2 and 37 pmole of ¹⁴ C-
NAD (0.06 uCi), 0.25-1.25 μg
PE and buffer (40 mM DDT, 1 mM EDT)
A, 50 mM Tris, pH 8.1). The activity was pm of NAD transferred to EF-2 in 30 minutes.
It is measured as an ole number. A standard curve for PE of known concentration has been established and this standard curve is used to determine the PE activity of the E. coli extract. After incubation at 37 ° C. for 30 minutes, 0.5 ml, 12% TCA is added to each quantitation mixture. The mixture for quantification is then
The mixture was ice-bathed for 5 minutes, followed by centrifugation at 3000 xg for 10 minutes at 4 ° C. The precipitate was washed with 1 ml of 6% TCA and centrifuged similarly. Thereafter, the ¹⁴ C radioactivity of the precipitate was measured with a liquid scintillation counter to obtain an index of ADP ribosylation activity.

Cytotoxicity test The cytotoxicity test of the modified PE is carried out using the NIH 3T3 cell line and human KB cells. NIH 3T3
The cells or human KB cells are isolated before the cytotoxic activity test.
The plate is allowed to stand in a four-compartment tissue culture plate at a density of 2 × 10 ⁴ cells per compartment for 24 hours. Different concentrations of PE or
B with plasmids expressing PEs of different sizes
The cells were incubated with the protein extract separated from L21 (DE ₃ ) for 48 hours, and the monolayer was stained with methylene blue to find out the surviving cells. The results are shown in Table 1.

Inhibition of Protein Synthesis Assay of protein synthesis inhibition by PE or by an extract obtained from B121 (DE ₃ ) / pJH8 and PE
Performed in the iss3T3 cell line. Swiss3
T3 cells are seeded one day before quantification in a culture vessel at a density of 10 ⁵ cells per compartment in a 24-compartment tissue culture plate. Cells are either PE (100 ng / ml) or differently sized P
PE (100) with extract containing excess E (Table 2)
ng / ml) before adding DMEM with 0.2 M
Washed once by replacing with% BSA.

After 15 minutes at 37 ° C., the medium is removed and replaced with fresh DMEM and 0.2% BSA. Four hours later, [ ³ H] leucine (final concentration 2-4 uCi / ml) is added to the medium for 1 hour, and the protein synthesis ratio is measured.

In a preferred embodiment of the present invention, as described above, the structural domain of the Pseudomonas exotoxin gene is incorporated downstream of the ribosome binding region of the T7 expression vector, with an ATG initiation codon (see FIG. 1). . 37 ° C.
And grown to A ₆₅₀ = 0.3. 1 mM IPTG was added to induce T7 RNA polymerase and 2
Incubated over time. As shown in Example 1, a series of plasmids is produced.

Next, a clone (pJH4) encoding a PE molecule having no leader sequence was constructed in which methionine was arranged close to the amino-terminal alanine in the processed natural PE (Table 1). The protein produced by pJH4 is designated as Met-PE. Large amounts of Met-PE, accounting for 20% of total cellular proteins, are produced by induction with IPTG. ADP ribosylation activity corresponding to 0.1 mg of native PE was observed in the supernatant. ADP ribosylation activity corresponding to 0.2 mg of natural PE per 1 mg of total cell protein was observed in the precipitate. p
PE molecules produced by JH4 differ from native PE molecules in that there is one extra methionine residue at the amino terminus.

As described above, pJH8 produced by pJH4 expresses a protein from which most of domain Ia has been deleted (only methionine added and only three amino acids at the amino terminus are retained at the amino terminus).

By immunoblotting, this protein was found to be 45 k
In b, the concentration was found to be approximately 0.04 mg / mg cell protein. Excluding urea and DTT from the reaction mixture, it shows high ADP ribosylation activity. Addition of urea and DTT to PJH8 reduces ADP ribosylation activity (about 30%), as opposed to native PE (addition of urea and DTT shows high ADP ribosylation activity).

Since the ADP ribosylation activity of PE is due to the carboxyl terminus of the molecule, a plasmid pJH17 composed of pJH4 (as described above) is produced. This plasmid contains 20 amino acids of domain III and domain Ib. The extract from this plasmid
Contains high ADP-ribosylation activity corresponding to 0.06 mg PE (Table 1). By immunoblotting, 31
kd protein was detected. This protein has a concentration of 0.03 m
Present at a concentration of g / mg cell protein.

Plasmids produced by the above method (and described in later examples) are pJH1, pJH2, pJH2,
Except for H4, none show significant cell destruction ability.
However, many of them show high enzymatic activity (first
table). These plasmids prevent inhibition of protein synthesis or cell destruction by native PE by exposing cells to 0.1 μg / ml native PE for 15 minutes in the presence of 3-5 μg / ml of various modified toxins. Is shown. The data in Table 3 show that plasmids expressing either domain Ia alone, or domain I, half of domain II and domain III, prevent protein synthesis inhibition of PE.

Animal toxicity Mice given native PE die from liver injury. For a 20 gram mouse, the lethal dose is 0.1-0.2 μg. Table 4 shows PEs lacking domain Ia.
Indicates that it significantly reduces toxicity to mice. Mice were injected intraperitoneally with native PE or native PE lacking domain Ia. Natural PE
All mice receiving 1.0 μg, 0.2 μg
Of mice that had been given PE died in 40 hours. Two of the three mice receiving 50 μg of PE Ia died. All mice receiving 20 μg of PE Ia survived. All mice that received 5 μg of PE Ia also survived. Thus, PE Ia was less than 100 times less toxic per body weight than native PE.

Gene Fusion Using Recombinant Pseudomonas Exotoxin Modified Among the PE genes for the modified PE of the present invention, particularly p gene
The gene contained in JH8 was converted to human α transforming growth factor (a-TGF) or human interleukin 2 (IL-
2) was fused to a DNA sequence encoding the same. Examples 7 and 8
Shows details of these gene fusions. The use of the modified PE is suitable for fusion with a peptide hormone, growth factor, or any other polypeptide cell recognition protein whose cell surface specific receptor is present on the cell surface. Some examples include insulin, glucagon, endorphin, growth hormone, melanocyte stimulating hormone, transferrin, bombesin, low-density lipoprotein, luteinizing hormone.
e) contains asialoglycoprotein.

EXAMPLES Example 1 As shown in Table 1, the process of the present invention was used to construct several plasmids containing fusions of different sized PE structural genes and the T7 late promoter. pJH2 contains an intact PE structural gene with an anterior fragment encoding a modified leader sequence. p
JH4 contains an intact PE structural gene with a methionine codon added to the amino terminus. pJH7 contains a gene encoding the structural domain III of PE (amino acids 405-613). pJH8 contains the genes encoding the structural domains II, Ib and III of pE (amino acids 253-613). pJH13 encodes a PE in which the first half of structural domain II, including amino acids 253-307, has been deleted. PJH14 encodes the structural domain Ia of PE (amino acids 1-252). pJH17 encodes structural domain III (amino acids 385-613) with an additional contiguous sequence of 20 amino acids from structural domain Ib.

PJH1 converts natural PE (pE0) to BamH
I was partially cut, and the linear DNA was eluted and completely cut with EcoRI. A 2.0 kb DNA fragment derived from pE0 has B
It has sites for amHI and EcoRI, and BamHI and EcoRI.
It was inserted into pAR2156 which had been completely cut with coRI.

PJH2 was constructed by partially cutting pJH1 with BamHI. The linear DNA was separated and completely cut with NdeI. 610 kb of D
The NA fragment was conserved for ligation with the synthetic oligonucleotide duplex below to construct pJH2. 5'TATGAAAAAAACTTTTTCTAG5 '

PJH4 was constructed by partially cutting pJH2 with TaqI. Linear DNA (6
10 kb) was isolated and completely cut with NdeI. The largest DNA fragment (5.9 kb) was isolated and ligated with the synthetic oligonucleotide heavy chain described below. 5 'TATGGCCGAAGAAGCTTT ACCGGCTTCTCTCAAAAGC5' This contains a HindIII site ... AAGCTT TTCGAA

PJH7 was constructed by partially cutting pJH4 with AatII. Linear DNA
(5.9 kb) was isolated and completely cut with HindIII. After separation, a 4.7 kb DNA fragment with AatII and HindIII sites was removed by S1 to remove attached proteins.
Nucleolytic enzymes were added and incubated, followed by ligation with T4 ligase.

PJH8 is constructed as described in the Detailed Disclosure of the Invention.

PJH13 was constructed by partially cutting pJH4 with AvaI. The linear DNA was separated and completely cut with EcoRI. AvaI on both ends
A 4.7 kb DNA fragment having the cleaved ends of E. coli and EcoRI was incubated with S1 to remove the cohesive end (DNA fragment 1). pJH4 was partially cut with SalI. Next, the linearized DNA fragment was subjected to EcoR
I cut completely. A 1.0 kb DNA fragment having SalI and EcoRI sites at the ends was digested with Klenow.
Incubation with DNA polymerase I and dNTPs filled in the cohesive ends (DNA fragment 2). DNA fragment 1
(4.7 kb) and DNA fragment 2 (1.0 kb) were ligated overnight at 4 ° C.

PJH14 was constructed by partially cutting pJH4 with AvaI. The linear DNA was completely cut with EcoRI. A 4.7 kb DNA fragment having AvaI and EcoRI sites at the ends was incubated with S1 to fill in the cohesive ends, and then ligated with T4 ligase.

PJH17 is constructed as described in the detailed description of the invention.

Example 2 The amount and activity of the recombinant toxin (produced by the plasmid described in Example 1) was determined by SDS-AGE, ADP ribosylation and cytocidal activity. The results are summarized first.

[0042]

[Table 1]

Example 3 An experiment was performed to test the properties of the recombinant toxin of the invention. The results are summarized in Table 2.

[0044]

[Table 2]

Example 4 The effect of various deletions on cellular protein synthesis in the presence or absence of native PE was determined. The results are shown in Table 3.
Structural domains Ia (pJH14) and half of structural domains I and II and III (pJH13) were extracted from sonicated cell pellets with 8M urea. 10 μl of each extract, comparable to 3-5 μg of recombinant protein, was used for the assay. Structural domains II, Ib, III (pJH
8) was present in the supernatant of sonicated BL21 (DE ₃ ) / pJH8 cells. 10 μl of extract comparable to 2 μg of recombinant toxin was used. As shown in Table 3, the cells were present at 0.
In the presence or absence of 1 μg / ml of natural PE
Minutes, then washed with 1 ml DMEM, DM
Incubate with EM in 0.2% BSA for 4 hours,
Then incubated with ³ H leucine for 1 hour.

[0046]

[Table 3]

Example 5 As shown in Table 4, Balb / c mice were challenged with various amounts of PE or PEIa in 1.0 ml of sterile saline and 10 mg / ml of sterile human albumin. Was determined intraperitoneally to determine the lethal dose in mice. Animals were observed daily for two weeks. All deaths occurred in 48 hours.

[0048]

[Table 4]

Example 6 As shown in FIGS. 3 and 4, an immunotokin consisting of a ₄₅ kD protein produced by binding pJH8 to an antibody against the human transferrin receptor (PE ₄₅ -HB21) via a disulfide bond was destroy human cells expressing the transferrin receptor (ID ₅₀ 3ng / ml). The above-mentioned immunotoxin has little or no effect on mouse cells not expressing human transferrin receptor even at 1000 ng / ml. In contrast, disulfide bonds cause HB
Natural PE bound to 21 has an ID _{50 of} 29 ng / ml.
To destroy mouse cells nonspecifically. FIG. 3 and FIG.
Data figures, non-specific toxicity of PE ₄₅ -HB21 is PE
−100% less than HB21.

Subsequently, the molecular weight of PE ₄₅ was measured again. The molecular weight was found to be 40,000.

Example 7 Construction of the α-transforming growth factor PE fusion gene To construct the small plasmid pVC8, which has few AvaII sites, pJH8 was transformed with Tth111
Treated with I and SphI. To create pVC31, pVC8 was partially cut with AvaII and ligated to a synthetic oligonucleotide (30 bp) containing a STuI site, a Tth111I site and a stop codon. pVC31
Was cut with Tth111I, filled in using a Klenow fragment which is a DNA polymerase fragment, and ligated to a blunt-end clone containing the α-TGF gene (pvC33). α-TGF gene, P-hTGF-10-925
[Derrynck et al, cells, 38: 287-2.
97 (1984)] was cut with EcoRI and BglI to obtain 3
A 22 bp fragment was obtained, separated, cut with Fnu 4HI, and
Treatment with 4 polymerase yielded a 152 bp fragment, which was then ligated to PVC31 to produce the PE-αTGF fusion gene. When expressed in E. coli, this plasmid produced an antibody to α-TGF and a protein with a molecular weight of 51,000 that reacted with PE.

Example 8 Construction of IL-2-PE fusion gene pVC8 was cleaved at 1190 by AvaII, a 3.6 fragment was separated, and its single-stranded end was Kleno.
The fragment I was obtained by filling with the enzyme w and dephosphorization. IL
To obtain one 1 kD fragment, clone PST-5
[Ga110 et al., PNAS, (81: 2543-254)
7 (1984)] is cut with PstI to obtain 1k of IL-2.
D fragment was obtained, which fragment was then placed at 105 and 669 at B
Cleaved with sp1286 and treated with T4 polymerase to fill in ends. The 564 bp fragment was ligated to fragment I and PHL-
Was prepared and transformed into BL21 expressing cells. By induction, it reacts with antibodies against IL-2 and PE and
A 60 kD protein with P-ribosylation activity was produced.

Indication of the deposit:
vil1e, American T of Maryland
type Culture Collection, deposited with an ATCC number, respectively, prior to the filing of this application. Also, when this application is patented, it will be maintained for 30 years from the date of the deposit or five years after the last deposit application, or for the longest of the term of the patent.
If the strain mutates or becomes nonviable during the deposit, it will be replaced. pJH12 ATCC 67205 pJHL-1 ATCC 67206 pVC33 ATCC 67207 pJH8 ATCC 67208 pJH14 ATCC 67209

[Brief description of the drawings]

FIG. 1 shows the construction of a plasmid of the invention used for the expression of different domains of PE.

FIG. 2 is a simplified map of different sized PE molecules produced as part of the present invention.

FIG. 3 shows the effect of PE ₄₅ -HB21 and PE-HB21 on protein synthesis in human KB cells. PE ₄₅ is a toxin protein lacking domain I encoded by plasmid pJH8. Cells are incubated with the appropriate immunotoxin for 24 hours and ³ H
-Incubated for 1 hour with leucine. The incorporation of ³ H-leucine into the protein was measured. Panel 3B
In, Swiss3T3 cells naturally Pseudomonas toxin (PE), native pseudomonas toxin coupled to HB21, or were incubated either with 24 hours of PE ₄₅ coupled with PE ₄₅ only or HB21. Cells were exposed to these drugs for 24 hours and protein synthesis was measured as described above.

FIG. 4 shows the effect of PE ₄₅ -HB21 and PE-HB21 on protein synthesis in human KB cells. PE ₄₅ is a toxin protein lacking domain I encoded by plasmid pJH8. Cells are incubated with the appropriate immunotoxin for 24 hours and ³ H
-Incubated for 1 hour with leucine. The incorporation of ³ H-leucine into the protein was measured. Panel 3B
In, Swiss3T3 cells naturally Pseudomonas toxin (PE), native pseudomonas toxin coupled to HB21, or were incubated either with 24 hours of PE ₄₅ coupled with PE ₄₅ only or HB21. Cells were exposed to these drugs for 24 hours and protein synthesis was measured as described above.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification FI A61K 39/02 ADZ A61K 37/24 (C12P 21/02 C12R 1:19) (71) Applicant 597072800 Sankar Adhya Sankar ADHYA United States of America 1878 Kings Grant Street, Geysersburg, MD 14400 (72) Inventor Ira Etchi Pastan Biol Mountain Road, Potomac, 20854 Maryland, United States 11710 (72) Inventor David Jay Fitzgerald 20902 Silver Spring, Rad Street, Maryland, United States 1731 (72) Inventor Sankar Adher 20878 Geysersba, Maryland, United States Kings Grant Street 14400

Claims (12)

[Claims] 1. A DNA encoding a modified Pseudomonas exotoxin having ADP ribosylation activity and the ability to translocate through cell membranes
Wherein the modified exotoxin is administered in vivo with lower toxicity to human or animal cells as compared to unmodified Pseudomonas exotoxin. With a modification of the receptor binding domain Ia of the native toxin sufficient to result in a modified toxin with lower toxicity to the liver when done. 2. An expression plasmid containing DNA encoding a complex containing a modified Pseudomonas exotoxin having ADP ribosylation activity and the ability to translocate through the cell membrane,
The modified exotoxin is less toxic to human or animal cells in vitro compared to unmodified Pseudomonas exotoxin fused to a targeting carrier, and when administered in vivo. Sufficient receptor binding domain Ia of the native toxin to result in a modified toxin of lower toxicity to the liver
Wherein the complex binds to a receptor or antigen on the target cell membrane. 3. The expression plasmid according to claim 2, wherein said targeting carrier is selected from the group consisting of antibodies, hormones, growth factors, cytokines, or other cell recognition proteins. 4. The expression plasmid according to claim 2, wherein said targeting carrier is an antibody. 5. The plasmid according to claim 3, wherein:
The growth factor is TFG-α. 6. The plasmid according to claim 3, wherein
The above-mentioned cytokine is interleukin-2. 7. The plasmid according to claim 4, wherein
The above-mentioned antibody is an anti-transferrin receptor antibody. 8. A modification comprising transfecting a suitable host cell with the plasmid of claim 1 and culturing said host cell under conditions such that a modified Pseudomonas exotoxin is produced. For producing a modified Pseudomonas exotoxin. 9. The method of claim 8, wherein the plasmid is selected from the group consisting of ATCC accession numbers 67206, 67207, and 67208. 10. A host cell containing the plasmid according to claim 1. 11. A targeting carrier comprising transfecting a suitable host cell with the plasmid of claim 2 and culturing said host cell under conditions such that said complex is produced. A method for producing a fused, modified Pseudomonas exotoxin. 12. A method for producing a modified Pseudomonas exotoxin fused to a targeting carrier, comprising: (a) transfecting a suitable host cell with the plasmid of claim 1; (b) Culturing the host cell under conditions such that a modified exotoxin is produced; and (c)
Complexing the modified exotoxin with a targeting carrier to produce a modified Pseudomonas exotoxin fused to the targeting carrier.